Zeolites for delivery of nitric oxide

ABSTRACT

There is described zeolites containing releasably adsorbed nitric oxide, methods of preparing the zeolites, methods of releasing the nitric oxide into a solution or into air and uses of the zeolites in therapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/562,401, which claims priority under 35 U.S.C. §371 from PCTApplication No. PCT/GB04/002905, filed in English on Jul. 5, 2004, whichclaims the benefit of Great Britain Application No. 0315540.5 filed onJul. 2, 2003, and Great Britain Application No. 0327222.6 filed on Nov.22, 2003, the disclosures and contents of which are incorporated byreference herein in their entireties.

FIELD OF INVENTION

The present invention relates to zeolites containing releasably adsorbednitric oxide, methods of preparing these zeolites, methods of releasingthe nitric oxide into a solution or into air, and uses thereof.

BACKGROUND OF THE INVENTION

Nitric oxide (the chemical formula is NO) is a remarkable small moleculethat is vitally important in many biological processes. It is avasodilator that increases blood flow through arteries and veins, and isalso an important factor in controlling/preventing platelet adhesion andaggregation and thrombus formation. It also plays a crucial role in theimmune system and in neurotransmission. Much is now known about the modeof action of nitric oxide and it is clear that it has enormous potentialin medicine and biotechnology in both in vivo and ex vivo applications.

The controlled delivery of nitric oxide may be important in prophylacticand therapeutic procedures and application. For example, nitric oxidecan prevent thrombosis and restenosis following balloon angioplasty andstent insertion in blocked arteries (International Patent Application WO95/24908). Because nitric oxide is active in many biological processes,targeted delivery is desirable however. The delivery of nitric oxide tothe skin may also have therapeutic benefits for patients with peripheralcirculatory problems which can occur in conditions such as arthritis andRaynaud's syndrome. Nitric oxide also exhibits antibacterial propertiesand incorporation into antibacterial devices and use for the treatmentof bacterial infections is desirable. Nitric oxide also plays a part inwound healing and angiogenesis, and delivery of nitric oxide to woundsand ulcers can be beneficial when healing is slow which can occur, forexample, in elderly patients (M. Shabani et al, Enhancement of woundrepair with a topically applied nitric oxide-releasing polymer Woundrepair and regeneration, 4, 353, 1996 and S. Frank H. Kampfer, C.Wetzler, J. Pfeilschifer, Nitric oxide drives skin repair: Novelfunctions of an established mediator Kidney International, 61, 882,2002).

However the delivery of nitric oxide to the desired area, and in therequired optimum dose is often difficult because nitric oxide is a gas.Delivery of nitric oxide is difficult in both ex vivo e.g. biotechnologyapplications and in vivo e.g. medical applications.

Various methods of nitric oxide delivery are known such as

-   -   (a) molecules which release NO spontaneously;    -   (b) molecules which are metabolised to give NO;    -   (c) molecules that release NO on photoactivation;    -   (d) release of NO from polymers and polymer coatings;    -   (e) production of NO from a chemical reaction.

The class (a) molecules include molecules known as nitric oxidenucleophile complexes (NONOates) (C. M. Maragos et al, Complexes of NOwith nucleophiles as agents for the controlled biological release ofnitric-oxide-vasorelaxant effects J. Med. Chem., 34, 3242, 1991). Theseare a variety of molecules which give off nitric oxide spontaneously andhave been shown to have a possible use in therapeutic applications (U.S.Pat. No. 4,954,526). However the use of NONOates in therapy is limitedbecause they become distributed throughout the body which may compromiseselectivity. The by-products following the release of NO may also formcarcinogenic secondary nitrosamines. Other class(a) molecules includenitrosothiols (Megson, I. L., Greig, I. R., Butler, A. R., Gray, G. A. &Webb, D. J. Therapeutic potential of S-nitrosothiols as nitric oxidedonor drugs Scot. Med. J. 42, 88, (1997)). Also, the class (a) moleculesmay cause dangerous lowering of systemic blood pressure.

The class (b) molecules include glyceryl trinitrate and sodiumnitroprusside (L. J. Ignarro Biosynthesis and metabolism ofendothelium-derived nitric-oxide Ann. Rev. Pharmacol. Toxicol. 30, 535,1990). These compounds are currently widely used as vasodilators,however prolonged use can lead to toxic side products such as cyanides.Furthermore, tolerance can be displayed because these molecules need tobe metabolised to release NO. The targeting of NO to particular sitesmay also be poor resulting in the effects tending to be systemic.

The class (c) molecules require specific activation, for example, lighthaving a specific wavelength which can be difficult to initiate (C.Works, C. J. Jocher, G. D. Bart, X. Bu, P. C. Ford, Photochemical NitricOxide Precursors Inorg. Chem., 41, 3728, 2002).

Class (d) release of nitric oxide mitigates the problems associated withsystemic activity by delivering nitric oxide to a specific target siteby supporting a nitric oxide releasing compound on a solid article. SuchNO releasing compounds may be polymeric materials which can be coatedonto medical instruments which can be used to target specific areas ofthe body for treatment. The polymers may contain, for example, the N₂O₂group that releases NO after a chemical reaction (International PatentApplication WO 95/24908 and US Patent Application 2002094985). However,the release of NO in such circumstances can be difficult to control andcurrently the preparation of the required materials may be expensive,often requiring multi-step processes and presenting difficult storageproblems because of instability at room temperature and require storageat a cold temperature. The possible use of such polymers has been shownin the treatment of cardiovascular problems, for example, restenosis;the manufacture of anti-thrombogenic medical devices; alleviation ofabnormal vasconstriction in the blood supply of the skin (Reynaud'ssyndrome) and for wound healing.

Class (e) delivery of nitric oxide has been proposed for topicalapplications by releasing nitric oxide from a chemical reaction. Thechemical reaction involves the application of sodium nitrite, ascorbicacid and maleic acid, which gives off NO when contacted by water (U.S.Pat. No. 6,103,275). However, this reaction takes place only in acidicconditions and so may cause irritation, especially to sensitive skin ofelderly patients. Additionally, the nitric oxide is released as ashort-lived burst rather than in a controlled manner.

The object of the present invention is to obviate and/or mitigate theproblems of nitric oxide storage and delivery.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided azeolite material comprising releasably adsorbed nitric oxide.

Zeolite materials are a class of aluminosilicate materials which areknown and used in a number of applications, for example, ion exchange,gas separation and catalysis (A. Dyer, An Introduction to ZeoliteMolecular Sieves, J. Wiley and Sons, 1988).

According to a second aspect of the present invention there is provideda method of preparing a zeolite material which comprises releasablyadsorbed nitric oxide, said method comprising the steps of providing azeolite material and contacting said material with nitric oxide gas.

Zeolites which are suitable for the present invention may be eithernaturally found or synthetically made. The zeolites contain pores andchannels having dimensions which allow small molecules or ions to beadsorbed onto the internal surfaces of the material. The general formulaof a zeolite framework is Al_(y)Si_(1-y)O₄ ^(y−). For each aluminiumatom in the zeolite framework, one negative charge is introduced whichmust be balanced by an extra-framework cation. These cations can beinorganic or organic in nature, and can be exchanged using standard ionexchange processes (M. E. Davis, Ordered porous materials for emergingapplications Nature 417, 813, 2002).

The zeolites may comprise transition element cations as theextra-framework species e.g. iron, copper, ruthenium, and such zeolitescan adsorb nitric oxide to form complexes inside the cavities of thezeolite material. These complexes are strong and may enable the nitricoxide to be stored until needed. Cations of other elements, for example,sodium and potassium bind nitric oxide less strongly. Those skilled inthe art may use standard ion exchange processes to introduce therequired metal ions into a zeolite structure as extra-framework cations(Plank et. al., U.S. Pat. No. 3,140,249; Preparation, characterisation,and Performance of Fe-ZSM-5 Catalysts R. Joyner and M. Stockenhuber, J.Phys. Chem. B., 1999, 103, 5963-5976). Using such techniques it ispossible to incorporate mixtures of cations in the zeolite structures.

The zeolites may be provided in a dehydrated state.

The amount of nitric oxide which may be loaded into the zeolites can becontrolled by varying the relative amounts of the extra-frameworkcations, controlling their chemical nature, and/or the total number ofions present. For example, the number of extra framework cations presentin the zeolite structure may depend on the amount of aluminium presentin the framework. More aluminium ions require more extra frameworkcations to balance the negative charge. The extra framework cations maythen interact with the NO molecules.

The chemical nature of the extra framework cations may also be changed(for example monovalent cations, e.g. Na⁺ and Ag⁺ may be exchanged fordivalent cations, e.g. Fe²⁺ and Cu²⁺ or trivalent cations, e.g. Ru³⁺ andFe³⁺). Each different cation may have a different affinity for NO andchanging the cations present in the zeolite framework may be used tocontrol the release of NO. Such manipulation of the zeolite compositioncan affect the rate at which the nitric oxide is released from thezeolite. For example a sodium-loaded zeolite may bind nitric oxide lessstrongly than an iron-loaded zeolite to release the nitric oxide morerapidly. A mixed sodium/iron zeolite may release nitric oxide at adifferent rate to either a sodium-loaded zeolite or an iron-loadedzeolite, and such release of nitric oxide may present a different rateprofile.

The choice of zeolite framework can also be used to vary the loading andrelease rate of nitric oxide. Zeolite frameworks are available insynthetic materials with a variety of different structures, and suitableframeworks may be chosen that offer the desired properties for theapplication under consideration. For example, the pores and channels ina zeolite structure may be defined by the size of the pore or channelopenings. The zeolite with the structure LTA has openings defined by 8pore tetrahedral units (i.e. a ring of 8Si/Al atoms and 8 oxygen atoms).Zeolite MFI has a larger ring opening defined by 10 tetrahedral units,and FAU by an even larger pore opening of 12 tetrahedral units. Thedimensionality of the pores can also differ between zeolite frameworks.For example, some zeolites have channels that run in only one direction(one dimensional channel systems) while others have systems ofinteracting channels in two or three dimensions (2-dimensional and3-dimensional channel systems). The size, shape and dimensionality ofzeolites may affect the rates of diffusion and adsorption/desorption ofNO, and may be used to control the rate of release of NO from thezeolite in a particular application.

Thus, the composition of the zeolite material may be tailored to controlthe amount of nitric oxide loaded into the zeolite structure and/or therate at which the nitric oxide is released from the zeolite.

Such zeolite structures may be chosen from, but not limited to,frameworks having the following three letter framework codes: LTA, FAU,MFI, MOR, FER, BEA, PHI and SAS (See the International ZeoliteAssociation (IZA) Website, for details of how the codes relate to theframework structures of the zeolites). These three letter codes describethe framework architecture of the zeolites, that is, their structure,but do not describe the composition of the zeolite which may varywidely. The three letter codes are used as a nomenclature system forzeolites.

The zeolites which may be used in the present invention may have thefollowing general formula (I):[(M1^(n+))_(x/n)(M2^(p+))_(y/p) . . .(Mn^(q+))_(v/q)][Al_(z)Si_(2-z)O₄]  (I)wherein M1 and M2 . . . Mn are extra framework metal cations of elementsselected from the group consisting of Li, Na, K, Ca, Mg, Fe, Cu, Ru, Rh,Co, Ni, Zn and Ag.

-   x may range from zero to nz,-   y may range from zero to pz, and-   v may range from zero to qz,-   subject to the condition that x/n+Y/p+y/p+ . . . +v/p=z.-   z is the number of silicon atoms replaced by aluminium atoms in the    zeolite framework.-   n+, p+ and q+ are the charges of the extra framework metal cations,    and may individually take the values of +1, +2 or +3.-   M1 and M2 . . . Mn may also be chosen from small organic cations    such as N(R₁)_(a)(R₂)_(b) ⁺ wherein R₁ and R₂ are independently    selected from H, —CH₃, —CH₂CH₃, or —CH₂CH₂CH₃, and a and b are    independently 0, 1, 2, 3 or 4 such that a+b=4;

When M1 and/or M2 are small organic cations, NH⁺ ₄ is preferred.

The zeolites which preferably may be used in the present invention havethe following general formula (II):[(M1^(n+))_(x/n)(M2^(p+))_(y/p)] [Al_(z)Si_(2-z)O₄]  (II)

-   wherein M1 and M2 are as defined previously,-   x may range from zero to nz, and-   y may range from zero to pz, subject to the condition that    x/n+y/p=z.

Prior to nitric oxide loading, the zeolites for use in the presentinvention may be fully or partially dehydrated, for example, undervacuum to remove water from the zeolite channels. The resulting zeolitemay then be exposed to nitric oxide to load the zeolite.

Typically, the nitric oxide loading is performed at a temperature from−100° C. to 50° C.

The loading of nitric oxide may be performed with pure NO or with amixture of NO and a carrier gas such as an inert gas, for examplehelium, argon or other inert gas including mixtures thereof.

The loading is typically performed at a pressure above atmosphericpressure, for example from atmospheric pressure up to a pressure of 10bar.

The nitric oxide loaded zeolites may be sealed inside airtight packagingfor storage and transport purposes'.

Upon exposure of the nitric oxide loaded zeolite to moisture, forexample an aqueous environment such as water or blood, the nitric oxideis displaced from the metal complex inside the zeolite resulting inrelease of nitric oxide gas into the aqueous environment.

The nitric oxide may also be released from the nitric oxide loadedzeolite when placed in air.

The release of nitric oxide may occur at a variety of temperatures,however room temperature or body temperature is preferred.

The nitric oxide loaded zeolite may be prepared in the form of a powderor a monolith for use for example in topical therapeutic applications orin vitro applications such as delivery of specific amounts of NO to cellcultures. For example, a specific amount of NO may be loaded into azeolite and then, knowing the extent of release or release profile ofthe NO loaded zeolite, a precise amount of NO may be delivered to thecell culture. This principle may also be applied to other deliveryapplications of NO e.g. in therapeutic, cosmetic and/or hygiene,applications so that a specific amount or dose of NO may beadministered.

The monoliths may be formed by compression of a zeolite powder or bymixing a powdered zeolite with a suitable binder which is well known inthe manufacture of zeolite catalysts.

Suitable binders include, but are not limited to, ceramic binders e.g.silica or alumina, and polymeric binders, e.g. polysulfone, polyethene,PET, polystyrene polytetrafluoroethylene (PTFE) and other polymers.

Alternatively the zeolites may be provided as coatings on medicaldevices such as metallic or plastics medical devices. The coated devicesmay then be delivered to the locality where the nitric oxide isrequired. For example, a zeolite coated stent may be used to performballoon angioplasty and the release of nitric oxide under theseconditions may be used to reduce restenosis.

Typically, the zeolites are provided in a suitable form as discussedabove, and then loaded with nitric oxide ready for storage and used at alater time.

A powdered zeolite loaded with nitric oxide may be used in topicalapplications such as for wound dressing, and may be provided, forexample, in a bandage for application to a wound for release of thenitric oxide into the wound to aid healing. A zeolite provided as amonolith may be used e.g. for topical applications or, for example, forsuppository application in the treatment of severe constipation.

According to a third aspect of the present invention there is provided azeolite material comprising releasably adsorbed nitric oxide for use insurgery and/or therapy.

According to a fourth aspect of the present invention there is provideda pharmaceutical, neutraceutical or cosmetic preparation comprising azeolite material comprising releasably adsorbed nitric oxide togetherwith a pharmaceutical/neutraceutical/cosmetic carrier therefor.

The present invention also provides the use of a zeolite materialcomprising releasably adsorbed nitric oxide in the preparation of amedicament for use in the treatment or prophylaxis of disease.

Diseases or medical conditions which may be treated include infectionsof the skin, including dermatophyte fungi, leishmaniasis, molluscum andpapilloma virus, and mycobacterium infections. Further uses includetherapeutic applications in anti-neoplastic activities, immune responsemodification, treatment of Raynaud's disease, wound healing and skinpigment modification. Yet further uses include treatment of restonsis,psoriasis and eczema, and skin cancer (melanoma). Therapies for otherbacterial problems include the reduction of severe foot or body odourproblems, and in the treatment, of Methicillin Resistant StaphylococcusAureus infections.

According to a sixth aspect of the present invention there is provided,a medical article comprising a zeolite material.

The zeolite material of the medical article may be provided withoutnitric oxide loaded therein to allow loading with nitric oxide prior touse and/or storage of the medical device ready for subsequent use.

Alternatively, the zeolite material of the medical article may beprovided as a zeolite material comprising releasably adsorbed nitricoxide.

Suitable medical articles for use in the present invention includestents, catheters, wound dressings, bandages, self-adhesive plasters andpatches.

The beneficial properties of nitric oxide may be advantageously employedin cosmetic and personal hygiene applications.

According to a seventh aspect of the present invention, there isprovided use of a zeolite comprising releasably adsorbed nitric oxide incosmetic and/or personal hygiene applications.

For example the zeolites of the present invention which comprisereleasably adsorbed nitric oxide may be used in cosmetic preparations;deodorants; skin preparations such as anti-aging skin preparations andpreparations applied before, during or after hair removal by shaving orby application of depilatory preparations; hair preparations; depilatorypreparations and the like.

Accordingly, the present invention also provides, as an eighth aspect, acosmetic and/or personal hygiene product comprising a zeolite whichcomprises releasably adsorbed nitric oxide.

The present invention also provides, as a ninth aspect, a method ofreleasing nitric oxide comprising the steps of

-   -   (i) providing a zeolite material comprising releasably adsorbed        nitric oxide;    -   (ii) contacting said zeolite material with a medium into which        said nitric oxide is to be released.

Such release of nitric oxide is preferably achieved in a controlledmanner, for example, by providing a suitable zeolite material with anestablished controlled release profile.

The medium into which the nitric oxide is to be released may be simplyair surrounding the nitric oxide loaded zeolite, or may be, for example,an aqueous medium.

The release may be performed either inside an animal body, topically toan animal body or in non-body applications such as release into cellcultures.

The release may be performed at any suitable temperature, however roomor body temperature is preferred.

The method of releasing nitric oxide may be applied to the treatment ofhumans or animals and accordingly the present invention further providesas an tenth aspect a method of treatment or prophylaxis of an individualin need thereof comprising providing a zeolite comprising releasablyadsorbed nitric oxide and contacting said zeolite with said individual.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Reference in the examples is made to the following figures in which:

FIG. 1 shows the crystal structure of dehydrated Na-zeolite-A;

FIG. 2 is a graph showing the release profile of NO into the atmosphereaccording to Example 3.

FIG. 3 is a bar chart showing the release profile of NO into theatmosphere at different times according to Example 5.

FIG. 4 is a graph which shows the amount of dissolved NO concentrationin accordance with Example 6;

FIG. 5 is a graph showing the release profile of NO into an argon flowin accordance with Example 6;

FIG. 6 is a graph showing the release profile of NO from Co— and Mn—exchanged zeolite-A in accordance with Example 7;

FIG. 7 is a graph showing the release profile of NO from Co-LTA(A) andCo-LTA(ZK-4) zeolites in accordance with Example 7;

FIG. 8 is a graph showing the aggregometer response over time for theblood platelet aggregation experiments performed in accordance withExample 8; and

FIGS. 9A and 9B are photographs of bacterial cultures showing theantibacterial action of NO-containing zeolites in accordance withExample 9.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention shall now be described withreference to the following non-limiting examples in which:

Examples 1, 1a and 1b describe the preparation of ion-exchangedzeolites;

Examples 2, 2a and 2b describe the preparation of nitric oxide-loadedzeolites;

Example 3 describes the release of nitric oxide from a nitric oxideloaded zeolite into the atmosphere;

Example 4 describes the release of nitric oxide from a nitric oxideloaded zeolite into solution;

Example 5 describes the release of nitric oxide from an alternativenitric oxide loaded zeolite into the atmosphere;

Example 6 describes the quantification of nitric oxide in solution bydirect measurement;

Example 7 describes the release of NO from Co- and Mn- exchangedzeolite-A into wet and dry argon atmospheres;

Example 8 describes the inhibition of platelet aggregation by aNO-loaded zeolite/PTFE disk; and

Example 9 describes the antibacterial action of NO-containing zeolites.

EXAMPLE 1 Preparation of Ion-exchanged Zeolites

The synthesis of zeolites is well known to those with knowledge of theart, and ion exchange of the zeolites can be carried out by standardmethods (Plank et. al., U.S. Pat. No. 3,140,249; Preparation,characterisation, and Performance of Fe-ZSM-5 Catalysts R. Joyner and M.Stockenhuber, J. Phys. Chem. B., 1999, 103, 5963-5976). Theion-exchanged zeolite is then dehydrated under vacuum to remove water.Analysis of the zeolites is carried out using elemental analysis, X-raydiffraction and spectroscopic analysis.

EXAMPLE 1a

An example of the preparation of a dehydrated ion-exchanged zeolite isdescribed below.

The zeolite (MFT, 2 g) was placed in a 0.05 M solution of the metal ion(200 ml, distilled water) to be exchanged and stirred for 24 hours.Alternatively, with the same concentrations the exchange could becarried out under dry conditions in an inert atmosphere (argon) withsonication using methanol as a solvent. The products were recovered byfiltration/centrifuge.

The concentration of the metal ion solution and time for the exchangecan be varied to vary loading of the metal into the zeolite. Specificexamples of different metal ions that have been loaded into the zeolitesare given in Table 1.

Table 1—Elemental composition of ion-exchanged zeolites prepared usingthis methodology. The table shows a range of ion exchange behaviour fromvery low exchange in the case of iron up to over exchange in the case ofcopper. Initial composition of the zeolite —(NH₄)_(Z)[Al_(Z)Si_(2-Z)O₄]Where z=0.13333 (Si/Al=14)

Cation (M) Final Al/M ratio Fe ³⁺ 17.82 Ni ²⁺ 8.42 Co ²⁺ 3.84 Cu ²⁺ 1.50

EXAMPLE 1b

Zeolite-A (given the three letter framework code LTA) is a well knownmaterial to those skilled in the art, manufactured in greater than 1Mtonne amounts annually for use as a detergent builder and watersoftener. The structure of zeolite-A (Pluth, J. J. & Smith, J. V.Accurate redetermination of crystal structure of dehydrated zeolite A.Absence of near zero coordination of sodium. Refinement of silicon,aluminium-ordered superstructure J. Am. Chem. Soc., 102, 4704 (1980) andCheetham, A. K., Eddy, M. M., Jefferson, D. A. & Thomas, J. M. A studyof Si,Al ordering in thallium zeolite-a by powder neutron-diffractionNature, 299, 24, (1982)) consists of alternating SiO₄ and AlO₄tetrahedra that share corners to produce the open framework depicted inFIG. 1, with ion exchangeable cations residing in the channels of thestructure. In this case the ion exchangeable cations are shown as sodiumcations bound to the oxygen atoms of the framework and can be readilyexchanged with transition metal ions. For clarity only the Al—O and Si—Obonds are drawn. The structure labelling in FIG. 1 is as follows: Si=1,Al=5, sodium cations=10 and oxygen atoms=15., Zeolite-A is well knownfor its affinity to water, often being used (under the name MolecularSieve 3A, 4A or 5A) to dry solvents in organic chemistry.

Samples of zeolite-A were synthesised according to the procedure givenin the Verified Syntheses of Zeolitic Materials (Robson H. & Lillerud,K.P., Verified Syntheses of Zeolitic Materials (2^(nd) Revised Edition)International Zeolite Association, (2001). The following ion-exchangeprocedure was then used to replace the sodium ions in the as-made formwith various transition metal cations known to bind nitric oxidestrongly (Mn²⁺, Ni²⁺, Cu²⁺, Co²⁺) to give metal ion-exchanged zeolites.Typically, the as-synthesised sodium zeolite-A (5 g) was placed in a0.05 M solution of the metal acetate (400 ml, distilled water) andstirred for 24 hours. The products were recovered by filtration, washedwith distilled water (400 ml) and dried at 100° C. overnight. Elementalanalysis was carried out to determine the chemical composition of thezeolites using an Agilent 7500 Series ICP-MS spectrometer. Thetransition metal zeolite A samples were then dehydrated to remove water.

EXAMPLE 2 Preparation of No-loaded Zeolites

Nitric oxide can be produced in situ, or introduced from a cylinder.

EXAMPLE 2a

An example of the preparation of an NO-loaded zeolite is given below.

A 1M asorbic acid solution (200 ml) was degassed by bubbling argonthrough the solution with stirring. This was then added dropwise tosodium nitrite (˜5 g) which had been purged with argon for 30 minutes. Aslow flow of argon was used to carry the'produced nitric oxide throughhigh surface area potassium hydroxide to remove higher nitrogen oxides,and then through calcium sulfate to dry the gas stream, before beingallowed to flow through the ion-exchanged zeolite (e.g., Fe-loaded MFTzeolite ˜0.5 g) then finally through a bubbler.

The NO-loaded zeolite is then sealed inside the vessel and stored underthe Ar/NO atmosphere (e.g. inside a sealed Schlenk tube at roomtemperature) until required. The same method of NO-loading can be usedfor all the zeolites, independent of framework type and ion exchange.

EXAMPLE 2b

Another example of the preparation of NO-loaded zeolite is given below.

The ion-exchanged zeolite-A (˜0.3 g) prepared according to Example 1bwas dehydrated for 2 hours at 300° C. in vacuo (0.5 mm Hg). This wascooled to room temperature and exposed to approximately 3 atm of anitric oxide/helium gas mixture (10% NO, 90% He) for 10 minutes,evacuated and exposed again to 3 atm of nitric oxide. This was repeatedthree times.

For the measurement of NO-release, a flow of argon (either saturatedwith water vapour or taken directly from the gas cylinder, 5 ml min⁻¹)was passed over a known amount of the NO-loaded zeolite. The gas wasthen bubbled through phosphate buffered saline solution (pH 7.4, 10 ml)in which a previously calibrated nitric oxide electrode (World PrecisionInstruments, ISO-NO Mark II) was immersed. The concentration of NO wasmeasured over the course of several hours. All experiments were repeatedthree times and gave reproducible results.

EXAMPLE 3 Release of NO into the Atmosphere from NO-loaded Fe-MFIZeolite

Thermogravimmetric analysis coupled with mass spectroscopic analysis ofthe resultant gases was used to study the temperature dependence of theevolution of nitric oxide from the zeolite. The results are reproducedgraphically in FIG. 2 which shows the profile of weight loss (line A)and ion current (line B) for NO in a mass spectrometer versustemperature. NO-loaded Fe-MFI zeolite (0.010 g) was placed in a NetzchThermogravimmetric analyser coupled to a mass spectrometer. The samplewas heated to 300° C. at 10° min⁻¹ 48 hours under flowing air and thegases evolved analysed using mass spectrometry. The resultant traceindicated that the amount of NO released increases up to 130° C. beforeit begins to reduce. However, at −180° C. a sharp spike in NO productionis seen, coinciding with a phase transition in the zeolite sample(confirmed by differential scanning calorimetry). This is the well knownmonoclinic to orthorhombic phase transition that occurs in zeolite MFI.The phase transition temperature can be altered by careful choice of thesilicon to aluminium ratio of the starting zeolite, by controlling theion exchanged cation and amount, and by controlling the amount of NOloading. Thus property can then lead to a tailored NO release, by forexample, a heat pad applied to a wound healing bandage—at temperaturesbelow the phase transition NO release is slow, while above the phasetransition NO release is much enhanced. FIG. 1 shows the phasetransition at 180° C., but there are literature reports of phasetransition in zeolite MFI as low as −100° C. (H Morell, K Angermund, A RLewis, D H Brouwer, C A Fyfe, H Gies Structural investigation ofSilicate-I loaded with n-hexane by X-ray diffraction, Si-29 MAS NMR, andmolecular modeling. Chem. Mater. 14, 2192, 2002). The precise transitiontemperature depends on the composition of the zeolite and the loading ofNO. Other zeolites, such as FER also show phase transitions that can betailored in this way.

EXAMPLE 4 Release of No into Solution from No-loaded Fe-MFI Zeolite

Fe-MFI nitric oxide adsorbed sample (0.013 g) was placed in distilledwater (10.452 ml) was tested for nitrite (Quantofix Nitrite Sticks)which give a positive result with 20 mg/l NO₂. A sample of distilledwater was tested for nitrite (as a reference) which resulted in 0 mg/lNO₂. Nitrite is formed in solution from the reaction of NO with waterand oxygen and is therefore an indirect method for the measurement of NOin solution.

EXAMPLE 5 Release of NO into Atmosphere from NO-loaded Fe-ZSM-5

A small sample of NO-loaded Fe-ZSM-5 (0.010 g) was placed in a NetzchThermogravimmetric analyser coupled to a mass spectrometer. The samplewas heated to 37° C. for 48 hours under flowing air and the gasesevolved analysed using mass spectrometry. The resultant trace indicatedthat NO is slowly released from the zeolite at these temperatures intothe atmosphere. FIG. 3 shows the profile of NO released from the zeoliteat different times during the cycle. The bar chart shows ion current(from mass spectrometer) versus time for four molecules (H₂O, NO, NO₂and HNO₂) released from NO-loaded Fe-MFI. It can be clearly seen that NOis the most abundant gas given off at all times.

EXAMPLE 6 Quantification of NO in Solution by Direct Measurement Using aNitric Oxide Electrode

The present invention is directed in particular to the delivery of NOabove chronic wounds, as animal models have shown that topicalapplication of NO can significantly promote wound closure (Shabnai M.,Pulfer S. K., Bulgran J. P. & Smith, D. J. Enhancement of wound repairwith a topically applied nitric oxide-releasing polymer. Wound Rep.Regen. 4, 353, (1996)) and there is evidence that NO can be used totreat diabetic ulcers (Witte, M. B., Kiyama, T. & Barbul, A, Nitricoxide enhances experimental wound healing in diabetes Br. J. Surg., 89,1594, (2002)). A useful model for this is the release of NO into a moistatmosphere that is in contact with the liquid phase (phosphate bufferedsaline pH 7.4). The amount of nitric oxide absorbed by the solution isthen measured using a nitric oxide electrode.

The World Precision Instruments ISO-NO Mark II nitric oxide electrodewas calibrated using the titration method according to the proceduredescribed by World

Precision Instruments (ISO-NO Mark II Instruction Manual, WorldPrecision Instruments, 2002). The metal ion-exchanged zeolite withadsorbed nitric oxide was transferred into a glass tube and wet argon (5ml min⁻¹) was allowed to flow over it. This stream was then directed tobubble through a buffered solution (pH 7.4 at 37° C.) into which thenitric oxide electrode was immersed. Data on the release of nitric oxidewas then collected over several hours.

FIG. 4 shows the dissolved nitric oxide concentration (not normalisedfor mass of zeolite or degree of ion exchange) produced when threeNO-loaded zeolite samples are exposed to a flow of moist argon asdescribed above. The gas flow is then bubbled through the bufferedsolution and the nitric oxide concentration measured with time. Theexperiment measures the uptake of nitric oxide by the solution, andtakes no account of loss of nitric oxide that does not dissolve in theliquid. However, for many of the proposed applications (e.g. as awound-healing bandage) where release of nitric oxide is not directlyinto a solution, this experiment mimics the situation more closely thanwould release of the nitric oxide directly into a liquid.

The results illustrate that different nitric oxide-loaded zeolitematerials release NO in different ways. Zeolites with the LTA structurerelease their NO relatively quickly, while those based on the PHIframework release nitric oxide over a much longer timescale. It is notedthat the copper and iron ion exchanged LTA zeolites show similar releaseprofiles. The results do show in all cases that the concentration ofnitric oxide in the solution is of similar magnitude (nanomolar tomicromolar concentrations) to that found in many biological situations.

FIG. 5 shows the NO release profiles measured as described above using anitric oxide electrode for a number of transition metal exchangedzeolite-A samples in contact with an argon flow that has been saturatedwith water vapour. The control is a Co²⁺— exchanged zeolite that has notbeen exposed to nitric oxide. The electrode response results have beennormalised to give the concentration of NO in solution per mg of zeolitematerial. The order of how much NO is released for each different metalagrees well with the NO adsorption properties of transition metalzeolites in pressure swing adsorption studies (Aria, H & Machida, M.Removal of NO through sorption-desorption cycles over metal oxides andzeolites Catal. Today 22, 97, (1994)). Co-exchanged zeolites releasingthe most NO while the original sodium form of the zeolite releases theleast NO. It is noted that the copper-exchanged zeolite-A results seemanomalously low, but, without wishing to be bound by theory it isbelieved that this is because the zeolite is overexchanged, with morecopper ions in the channels than is strictly necessary for chargebalance reasons. Many of the ‘extra’ copper ions are probably present ashydroxide species (Yahior, H. & Iwamoto M. Copper ion-exchanged zeolitecatalysts in deNO(x) reactions Appl. Catal. A. 222, 163, (2001)) and soreduce the availability of the metal ions for NO coordination.

The cross-over of the Mn²⁺ and Ni²⁺ exchanged zeolites may indicatedifferent distributions of the metal ions between the three possibleextra framework cations sites in zeolite-A, some of which may be moresusceptible to substitution by water than others.

It is also noted that the release of NO takes place over a relativelylong period of time (about 10 hours in FIG. 5), and if there is lesswater vapour present, the release takes place over an even longer timeperiod.

EXAMPLE 7 NO Release of Co— and Mn— Exchanged Zeolite a into Both ‘Wet’and ‘Almost Dry’ Argon Atmospheres

FIG. 6 shows the release profile of Co- and Mn-exchanged zeolite A intoboth ‘wet’ (water vapour saturated) and ‘almost-dry’ argon atmospheres,and in the latter case the zeolites still gave off measurable amounts ofnitric oxide more than 24 hours after the experiment began. This showsthe importance of water in the mechanism of the NO release from thesezeolites.

In the ‘wet’ experiments the argon was bubbled through hot (80° C.)deionised water prior to contacting the zeolite. In the dry experimentthe argon was taken directly from the gas cylinder and partially driedover calcium sulphate.

The amount of nitric oxide released by the zeolite appears to depend notonly on which transition metal is present but also on how much of aparticular metal is present. Zeolite-ZK4 is a variant of zeolite-A thathas the same framework structure and so has the same framework code(LTA). However, there are fewer exchangeable cations in zeolite-ZK4 asthere is aluminium in the framework. This means that there are fewermetal cation sites in the channels of the structure to bind nitricoxide. It can be clearly seen in FIG. 7 that Co-exchanged zeolite-Areleases more NO than Co-exchanged zeolite ZK4, consistent with thereduced level of cobalt in the ZK4 structure.

The above experiments indicate the potential of NO-loaded zeolites todeliver nitric oxide into a moist atmosphere for delivery above the skinfor applications such as the promotion of wound healing, the treatmentof diabetic ulceration or the prevention of bacterial infection. Theyalso illustrate the controllable nature of the NO delivery, which can bechanged by varying the type and amount of transition metal present inthe zeolite structure.

EXAMPLE 8 Inhibition of Platelet Aggregation

There is a need for improvements in the biocompatibility of materialse.g. for blood contacting solids that are used in vascular grafts andextracorporeal tubing that is necessary in coronary bypass surgery.Life-threatening complications can occur if thrombosis formation(platelet aggregation and adhesion) is induced by materials that are incontact with blood (Keefer, L. K. Thwarting thrombus Nature Materials,2, 357, (2003)). Thrombus formation in healthy circulatory systems isinhibited in a number of ways, including the production of smallquantities (approximately 1 pmol min⁻² mm⁻²) of NO by the endothelialcells that line the blood vessels and by blood platelets.

A potentially important strategy for reducing post-operativecomplications is to provide medical devices comprising an NO-releasingzeolite in accordance with the present invention, thereby mimicking theaction of the endothelial cells. The Co-exchanged zeolite-A samplesprepared as described previously, in a 75:25 wt % mixture with powderedpolytetrafluoroethylene (PTFE) were prepared as mechanically stablepressed disks as follows.

The zeolite was ground with PTFE in the desired ratio (75% zeolite: 25%PTFE). The mixture was then pressed into disks (5 mm, ˜20 mg) under 2tons for 30 seconds.

The disks were then dehydrated and loaded with nitric oxide in the sameway as the powder samples. Tests with disks made from only NO-exposedPTFE showed no delivery of nitric oxide. The zeolite/PTFE disks werethen suspended in a steel wire holder below the surface of platelet richplasma (PRP) (prepared as described below) in the cuvette of afour-channel platelet aggregometer at 37° C. After a short inductionperiod (1 minute), platelet aggregation was initiated and then measuredas a change in turbidity (light transmission) of PRP against a plateletpoor plasma (PPP) blank. The results depicted as a graph in FIG. 8 showthat a NO-loaded Co-exchanged zeolite-A/PTFE sample completely inhibitsplatelet aggregation (line 2) while a Co-exchanged zeolite/PTFE samplethat has not been loaded with NO shows no inhibition of aggregation(line 3) when compared to a PRP control where no zeolite or PTFE wasadded (line 1). This experiment illustrates well the potential of theNO-loaded zeolite-A to inhibit thrombosis in physiological solutions andthe possibilities of using the zeolites as NO-releasing components inmedical devices, e.g. when blended with polymers such as PTFE.

Preparation of Platelet Rich Plasma

Venous blood was drawn from the antecubital fossa of healthy volunteers(aged 20-40 years) into citrated tubes (0.38% final concentration).Volunteers had not taken any medication known to affect plateletaggregation within the last 10 days. Platelet rich plasma (PRP) wasobtained from whole blood by centrifugation (350 g; 20 min; roomtemperature). Platelet poor plasma (PPP) was obtained by furthercentrifugation of PRP (1200 g; 5 min; room temperature).

EXAMPLE 9 Antibacterial Action of NO-containing Zeolites

Between 1 and 10 mg of NO-loaded cobalt exchanged zeolite-A powder wereplaced in the centre of bacterial cultures (P. aureginosa and E. coli)grown on agar. After 24 hours the area of bacteria killed was measured.The effectiveness of NO-containing zeolite-A was approximately 2 to 3times greater than that of C-zeolite-A that was not loaded with NO.

FIGS. 9a and 9b show the anti-bacterial effect of NO-loaded Co-exchangedzeolite-A as dark areas 25 around the power grams 20. The light areas 30are living bacterial culture. The bacteria used are P. aureginosa (FIG.9a ) and E. Coli (FIG. 9b ).

In summary, the present invention is shown to have application in NOstorage and release for biological and medical applications. Thepreparation and loading of zeolites with nitric oxide is relativelyfacile, and the NO loaded zeolites are stable when stored in anhydrousconditions at room temperature. The delivery of NO occurs by simplereaction with water, and the amount of nitric oxide released can betailored by altering both the type and number of metal cations in thezeolite structures. NO-releasing zeolites according to the presentinvention are shown to inhibit platelet aggregation in physiologicalfluids, a potentially important application in the prevention ofthrombus. The examples hereinabove are not to be construed as limitingon the scope of the present invention, but merely representativeembodiments thereof. Other ways of performing the invention will beapparent to the skilled person.

The invention claimed is:
 1. A method of releasing nitric oxide (NO) atbody or room temperature from a zeolites comprising extra-frameworkcations, the method comprising the steps of: (i) providing a partiallyor fully dehydrated aluminosilicate zeolites comprising extra-framworkcations selected from the group consisting of Fe, Cu, Ru, Rh, Co, Ni,Zn, and Ag, the extra-framework cations being effective to chemicallybind nitric oxide and having nitric oxide bound thereto, (ii) contactingthe zeolites from (i) with moisture at body or room temperature, wherebynitric oxide is displaced from the zeolites and released, wherein theproviding step comprises administering the zeolites to an animal subjecteither inside the animal subject or topically, and the contacting andrelease of nitric oxide occurs either inside the animal body ortopically, respectively.
 2. The method according to claim 1, wherein themoisture is comprised within blood of the animal subject.
 3. The methodof claim 1, wherein the providing step comprises topically administeringthe zeolites to an animal subject.
 4. The method according to claim 3,wherein the zeolites is administered in conjunction with a surgicalprocedure.
 5. The method according to claim 1, wherein the zeolites hasan LTA (Linde Type A/Zeolite A) framework structure.
 6. The methodaccording to claim 1, wherein the zeolites is in the form of a powder ora monolith.
 7. The method according to claim 6, wherein said monolith isformed by compression of a zeolites powder or comprises a powderedzeolites and a binder.
 8. The method according to claim 7, where thebinder is selected from ceramic binders and polymeric binders.
 9. Themethod according to claim 1 wherein the zeolites in step (i) is providedin sealed airtight packaging which is removed prior to use in thecontacting step.
 10. The method according to claim 1, wherein the animalsubject possesses a disease or medical condition selected from the groupconsisting of infection of the skin; Raynaud's disease; possession of awound; restenosis; psoriasis, eczema, skin cancer (melanoma); severefoot or body odor, and methicillin-resistant Staphylococcus aureusinfection.
 11. The method according to claim 1, wherein the zeolites instep (ii) is provided as part of a medical article selected from thegroup consisting of a stent, a catheter, a wound dressing, a bandage, aself-adhesive plaster and a patch.
 12. The method of claim 1, for use ina cosmetic or personal hygiene treatment of an individual, wherein step(i) comprises providing the zeolites in the form of a cosmeticpreparation, deodorant, depilatory, or hair preparation.
 13. The methodof claim 1, wherein the zeolites has a framework structure selected fromLTA (Linde Type A/zeolite A), FAU (faujasite), MFI (ZSM-5) MOR(mordenite), FER (ferrierite), BEA (zeolites beta), PHI (zeolites Phi)and SAS (STA-6/St. Andrews Six).
 14. The method of claim 1, wherein theproviding step comprises administering the zeolites to an animal subjectinside the animal subject.